Isoprene (2-methyl-1,3-butadiene) is the critical starting material for a variety of synthetic polymers, most notably synthetic rubbers. Isoprene is naturally produced by a variety of microbial, plant, and animal species. In particular, two pathways have been identified for the biosynthesis of isoprene: the mevalonate (MVA) pathway and the non-mevalonate (DXP) pathway. However, the yield of isoprene from naturally-occurring organisms is commercially unattractive. Isoprene can also be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming. Petroleum cracking of the C5 stream of hydrocarbons produces only about 15% isoprene. About 800,000 tons per year of cis-polyisoprene are produced from the polymerization of isoprene; most of this polyisoprene is used in the tire and rubber industry. Isoprene is also copolymerized for use as a synthetic elastomer in other products such as footwear, mechanical products, medical products, sporting goods, and latex.
Isoprenoids are compounds derived from the isoprenoid precursor molecules IPP and DMAPP. Over 29,000 isoprenoid compounds have been identified and new isoprenoids are being discovered each year. Isoprenoids can be isolated from natural products, such as microorganisms and species of plants that use isoprenoid precursor molecules as a basic building block to form the relatively complex structures of isoprenoids. Isoprenoids are vital to most living organisms and cells, providing a means to maintain cellular membrane fluidity and electron transport. In nature, isoprenoids function in roles as diverse as natural pesticides in plants to contributing to the scents associated with cinnamon, cloves, and ginger. Moreover, the pharmaceutical and chemical communities use isoprenoids as pharmaceuticals, nutraceuticals, flavoring agents, and agricultural pest control agents. Given their importance in biological systems and usefulness in a broad range of applications, isoprenoids have been the focus of much attention by scientists.
Methods for the production of isoprene, isoprenoid precursor molecules, and/or isoprenoids by recombinant cells at high rates, titers, and purities have been disclosed (see, for example, International Patent Application Publication No. WO 2009/076676 A2. However, industrial production of these molecules by engineered cells requires a fermentable feed stock carbon source, typically derived from an agriculturally-based starch, on which the cells can grow. A less expensive source of carbon used during the production process has the potential to lower the cost of industrial production for isoprene, isoprenoid precursor molecules, and/or isoprenoids.
A number of agricultural crops are viable candidates for the conversion of starch to fermentable feed stock. Such fermentable feedstocks can be fed to various microbes to produce a variety of biochemicals. Typically, corn is used as the primary starch source for producing fermentable glucose. However other high-starch content sources like sorghum, wheat, barley, rye and cassava are beginning to gain more attention as a viable feedstock for the industrial production of biochemicals and fuel. The conventional process for producing a fermentable high glucose syrup feedstock from insoluble starch involves heating whole ground grain or starch slurry to temperatures in excess of 95° C. in the presence of alpha amylase (a process known as “liquefaction”), followed by cooling, pH adjustment, and subsequent glucoamylase hydrolysis (otherwise known as “saccharification”). Such processes can produce fermentable feed stocks containing greater than 90% glucose. However, these conventional approaches are highly energy-intensive.
Various industrial processes have been adopted by the starch sweetener industry for enzyme-mediated liquefaction (see, e.g. U.S. Pat. No. 5,322,778). Some of these processes are performed at lower temperatures with relatively low steam requirements (e.g., 105-110° C. for 5-8 min) while others are high temperature processes (e.g., 148° C.+/−5° C. for 8-10 sec), resulting in improved gelatinization of starch granules leading to improved filtration characteristics and quality of the liquefied starch substrate (Shetty, et al., (1988) Cereal Foods World 33:929-934). Further advances in the liquefaction process have been demonstrated by multiple additions of thermostable alpha amylases in the pre/post jet cooking step, which results in significant improvements with respect to yield loss, processing costs, energy consumption, pH adjustments, temperature thresholds, calcium requirements and levels of retrograded starch.
The drastic conditions required for liquefaction (e.g. high temperature and pH), negatively affect the bioconversion efficiency of whole ground grains into feedstocks, resulting in the loss of fermentable sugars, production of Maillard reaction products, destruction of essential nutrients (e.g., free sugars, free amino acids, minerals, vitamins), deactivation of native beneficial enzymes (e.g. amylases, proteases, and phytases) and/or cross-linking or condensation of cellular components such as tannins and starch proteins (Wu et al., Cereal Chem., 2007, 84:130-13). Furthermore, significant energy costs are associated with high-temperature cooking of grain to aid in enzymatic digestion. In addition, in the context of the dry grind process for ethanol, the incomplete gelatinization during high temperature cooking (i.e. temperatures exceeding the starch gelatinization temperature) for solubilizing granular starch in ground whole grain is believed to be the principle reason for lower digestibility by alpha amylases. The digestibility of starch is also negatively affected by starch-lipid and starch-protein complexes formed during the interaction of reactive proteins and lipids with starch at liquefaction conditions (Zhang & Hamaker, Cereal Chem., 1998, 75:710-713). Another major problem associated with liquefaction at high temperature is high viscosity due to the rapid swelling of starch and non-starch polysaccharide components such as beta-glucan.
Due to increasing concern for the environment and the need to limit greenhouse gases, sources of renewable energy are gaining wide-spread attention. The recent development of no-cook processes using enzymes capable of hydrolyzing granular starch directly into fermentable glucose have made significant improvements in the energy required for ethanol production (see, e.g., U.S. Pat. No. 7,037,704; U.S. Patent Application Publication Nos.: 2003/0180900 A1, 2006/0121589 A1, and US 2004/0234649 A1; and International Patent Application Publication No.: 2004/081193 A2). However, these processes require extensive milling for fine grind particles, longer fermentation times, and potential risk of microbial infection.
Another major problem associated with current processes for the production of high fermentable glucose syrup (such as syrups with greater than 96% fermentable sugar) is loss of glucose yield due to the production of the reversion reaction product, isomaltose (6 O-α-D-glucopyranosyl-a [1-6]-α-D-Glucopyranoside). This reversion reaction can be catalyzed by glucoamylases during saccharification. Many microorganisms used for the production of valuable biochemicals cannot ferment isomaltose, and such approaches also result in downstream processing losses during recovery, and yield losses. What is needed, therefore, is a low temperature process for the direct production of fermentable feedstocks from ground or fractionated grain that avoids the drawbacks associated with currently available no-cook enzymatic starch hydrolysis processes which contains reduced amounts of reversion products and which can serve as a carbon source for the production of isoprene, isoprenoid precursor molecules, and/or isoprenoids.
The invention described herein addresses these problems and provides additional benefits as well.
All patents, patent applications, publications, documents, nucleotide and protein sequence database accession numbers, the sequences to which they refer, and articles cited herein are all incorporated herein by reference in their entireties.